The present invention resides in the technical fields of immunology and enzymology.
The cytochrome P450 family of enzymes is primarily responsible for the metabolism of xenobiotics such as drugs, carcinogens and environmental chemicals, as well as several classes of endobiotics such as steroids and prostaglandins. Members of the cytochrome P450 family are present in varying levels and their expression and activities are controlled by variables such as chemical environment, sex, developmental stage, nutrition and age.
More than 200 cytochrome P450 genes have been identified. There are multiple forms of these P450 and each of the individual forms exhibit degrees of specificity towards individual chemicals in the above classes of compounds. In some cases, a substrate, whether it be drug or carcinogen, is metabolized by more then one of the cytochromes P450.
Cytochrome P450 2C series of enzyme are of major importance in the use of drugs for the treatment of various disease conditions. The 2C family ranks among the most important of all of the P450 enzymes in humans.
Human cytochrome P450 2C9 is of major importance being responsible for the metabolism of numerous drugs and non-drug xenobiotics including tolbutamide, S-warfarin, phenytoin, diclofenac, ibuprofen, and losarten. The three major alleles of P450 2C9 are the wild type 2C9Arg144(*1), 2C9Cys144(*2), 2C9Ilexe2x86x92Leu359(*3) (Haining et al. 1996, Miners and Birkett 1998; hereinafter referred to as 2C9*1, 2C9*2 and 2C9*3 respectively). A genetic study of the 2C9 polymorphism indicated that the frequency of the 2C9* 1 and 2C9*3 alleles in a Caucasian American Population sere 0.08% and 0.06% respectively and 0.005 and 0.01% respectively in Afro-Americans (Sullivan-Klose et al. 1996). In a Japanese population the 2C9*2 allele was absent and the frequency of 2C9*3 was 0.021 (Nasu et al. 1997). In another study of 100 Caucasians the allelic frequency for the 2C9 wild type, 2C9*1, 2C9*2 and 2C9*3 were 0.79, 0.12 and 0.085 respectively (Stubbins et al. 1996).
The catalytic roles of the 2C9*1, 2C9*2 and 2C9*3 for the metabolism of warfarmn, flurbuprofen and diclofenac were studied in liver microsomes from 30 humans that were genotyped for the three 2C9 alleles. Nineteen of the humans were wild type, eight were heterozygous 2C9*2 and three were heterozygous for 2C9*3. All of the individuals with the 2C9*2 allele had similar but slightly lower activities for the metabolism of the three substrates. One of the three samples from heterozygotes for 2C9*3 had low Vmax and high Km while the other two samples were comparable to that of the 2C9*1 wild type and the 2C9*2. The conclusion of the study was that the 2C9*2 allele exhibited comparable but slightly lower metabolic activity than the wild type and the 2C9*3 had slower rates of oxidation compared to the wild type (Yamazaki et al. 1998). In another study (Veronese et al. 1991) found that the Vmax for the 2C9*2 allele for tolbutamide and phenytoin metabolism were 2-3 lower than the wild type 2C9. In a different study the Vmax of (S)7-hydroxy warfarmn formation was 20-fold greater in the wild type than the 2C9*2. The Vmax for methyihydroxy tolbutamide formation however was similar in the wild type and the 2C9*2 allelic (Rettie et al. 1994).
Typical substrates for 2C family members include taxol, tobutamide, pheytoin, lansoprazolem mephytoin, arachidonic acid, cyclophosphamide, ifosphamide, debrisoquine, methoxylflurane, tienilic acid, phenanthrene tolbutamide, benzo(a)pyrene, 58C80 (2-(4-t-Butylcyclohexhl)-3-hydroxyl-1,4-naphthoquinone), torsemide, aracidonic acid, mephenytoin, 1-tetrahydrocannabinol, and warfarin.
Genetic polymorphisms of cytochromes P450 result in phenotypically-distinct subpopulations that differ in their ability to perform biotransformations of particular drugs and other chemical compounds. These phenotypic distinctions have important implications for selection of drugs. For example, a drug that is safe when administered to most humans may cause toxic side-effects in an individual suffering from a defect in an enzyme required for detoxification of the drug. Alternatively, a drug that is effective in most humans may be ineffective in a particular subpopulation because of lack of a enzyme required for conversion of the drug to a metabolically active form. Further, individuals lacking a biotransformation enzyme are often susceptible to cancers from environmental chemicals due to inability to detoxify the chemicals. Eichelbaum et al., Toxicology Letters 64/65, 155-122 (1992). Accordingly, it is important to identify individuals who are deficient in a particular P450 enzyme, so that drugs known or suspected of being metabolized by the enzyme are not used, or used only with special precautions (e.g., reduced dosage, close monitoring) in such individuals. Identification of such individuals may indicate that such individuals be monitored for the onset of cancers.
Existing methods of identifying deficiencies in patients are not entirely satisfactory. Patient metabolic profiles are often assessed with a bioassay after a probe drug administration. Poor metabolizers (PM) exhibit physiologic accumulation of unmodified drug and have a high metabolic ratio of probe drug to metabolite. This bioassay has a number of limitations: Lack of patient cooperation, adverse reactions to probe drugs, and inaccuracy due to coadministration of other pharmacological agents or disease effects. See, e.g., Gonzalez et al., Clin. Pharmacokin. 26, 59-70 (1994). Genetic assays by RFLP (restriction fragment length polymorphism), ASO PCR (allele specific oligonucleotide hybridization to PCR products or PCR using mutant/wild-type specific oligo primers), SSCP (single stranded conformation polymorphism) and TGGE/DGGE (temperature or denaturing gradient gel electrophoresis), MDE (mutation detection electrophoresis) are time-consuming, technically demanding and limited in the number of gene mutation sites that can be tested at one time.
A complication in patient drug choice is that most drugs have not been characterized for their metabolism by P450 2C family and other cytochromes P450. Without knowing which cytochrome(s) P450 is/are responsible for metabolizing an individual drug, an assessment cannot be made for the adequacy of a patient""s P450 profile. For such drugs, there is a risk of adverse effects if the drugs are administered to poor metabolizers.
Monoclonal antibodies that specifically bind to 2C family members and inhibit its activity, if available, could be used to screen drugs for their metabolism by 2C and/or identify 2C poor metabolizers by simple bioassays, thereby overcoming the problems in prior complicated methods discussed above. However, such monoclonal antibodies represent, at best, a small subset of the total repertoire of antibodies to human cytochrome P450 2C, and have not hitherto been isolated. Although in polyclonal sera, many classes of antibody may contribute to inhibition of enzyme activity of P450 2C family members as a result of multiple antibodies in sera binding to the same molecule of enzyme, only a small percentage of these, if any, can inhibit as a monoclonal. A monoclonal antibody can inhibit only by binding in such a manner that it alone block or otherwise perturb the active site of an enzyme. The existence and representation of monoclonal antibodies with inhibitory properties thus depend on many unpredictable factors. Among them are the size of the active site in an enzyme, whether the active site is immunogenic, and whether there are any sites distil to the active site that can exert inhibition due to stearic effects of antibody binding. The only means of obtaining antibodies with inhibitory properties is to screen large numbers of hybridoma until one either isolates the desired antibody or abandons the task through failure.
Notwithstanding these difficulties, the present invention provides inter alia monoclonal antibodies that specifically bind to human cytochrome P450 2C family members and inhibit their activity.
The invention provides isolated binding agents that compete with a monoclonal antibody MAb 292-2-3 for specific binding to human cytochrome P450 allelic variant 2C9*2 without specifically binding to human cytochrome 2C9*1 and 2C9*3, and that specifically inhibits 2C-catalyzed metabolism of phenanthrene by at least 50%. The invention further provides other isolated binding agents that compete with a monoclonal antibody MAb 763-15-5 for specific binding to the human cytochrome p450 2C9 allelic variants 2C9*1, 2C9*2, and 2C9*3, and that specifically inhibits 2C-catalyzed metabolism of phenanthrene by at least 50%. The invention further provides isolated binding agents that compete with a monoclonal antibody MAb 763-15-20 for specific binding to the human cytochrome P450 2C9 allelic variants 2C9*1, 2C9*2, and 2C9*3. The invention further provides isolated binding agents that compete with a monoclonal antibody MAb 592-2-5 for specific binding to human cytochrome P450 2C9 and 2C18, and that specifically inhibits 2C-catalyzed metabolism of phenanthrene by at least 50%. The invention further provides isolated binding agents that compete with a monoclonal antibody MAb 5-7-5 for specific binding to a human cytochrome p450 2C family member selected from the group consisting of 2C9, 2C18, and 2C19, and that specifically inhibits 2C-catalyzed metabolism of phenanthrene by at least 50%.
Preferred binding agents are monoclonal antibodies. Some binding agents lack specific binding to at least one cytochrome P450 selected from the group consisting of human cytochromes P450 1A1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5. Some binding agents lack specific binding to each of human cytochromes P450 1A1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5. Preferred binding agents are able to specifically inhibit the enzyme activity of human cytochrome P450 allelic variant 2C9*2 by at least 90%. Other preferred binding agents are able to specifically inhibit the enzyme activity of human cytochrome P450 allelic variant 2C9*2 by at least 70%. Other preferred binding agents are able to specifically inhibit the enzyme actibity of human cytochrome P450 allelic variants 2C9*1 and 2C9*3 by at least 70%. Some binding agents are binding fragments, such as Fab fragments.
MAb 292-2-3, MAb 592-2-5, MAb 5-7-5, MAb 5-1-5, MAb 281-1-1, MAb 763-15-5, and MAb 763-15-20 are exemplified monoclonal antibodies. Some other monoclonal antibodies are analogs of these monoclonal antibodies comprising a light chain variable domain having at least 80% sequence identity with the light chain variable domain of a monoclonal antibody selected from the group consisting of MAb 292-2-3, MAb 592-2-5, MAb 5-7-5, MAb 5-1-5, MAb 281-1-1, MAb 763-15-5, and MAb 763-15-20, wherein the percentage sequence identity is determined by aligning amino acids in the light chain variable domains by the Kabat numbering convention and a heavy chain variable domain having at least 80% sequence identity with the heavy chain variable domain of a monoclonal antibody selected from the group, wherein the percentage sequence identity is determined by aligning amino acids in the heavy chain variable domains by the Kabat numbering convention.
The invention further provides cell lines producing monoclonal antibodies as described above. Cells lines can be eucaryotic or procaryotic.
The invention further provides methods of determining whether a cytochrome P450 2C family member metabolizes a compound. Such methods entail contacting the compound with cytochrome P450 2C family member in the presence of varying amounts of the binding agents above. Metabolism of the compound is then assayed as a function of amount of binding agent, a decrease of metabolism with amount of binding agent indicating that cytochrome P450 2C family member metabolizes the compound. In some such methods, the compound is contacted with cytochrome P450 2C family member in a sample containing a collection of cytochrome P450 enzymes including the 2C family member. Preferred P450 2C family members are P450 2C9*1, 2C9*2, 2C9*3, 2C8, 2C18, and 2C19.
In some methods, the sample is a tissue sample. In some methods, the collection of enzymes are obtained from a cell culture expressing the enzymes. In some methods, the compound is a drug, steroid or carcinogen.
The invention further provides methods of detecting cytochrome P450 2C members. Such methods entail contacting a sample suspected of containing cytochrome P450 2C family member with a binding agent described above. One then determines whether the agent specifically binds to the sample, specific binding indicating the presence of the particular cytochrome P450 2C family member in the sample.
The invention further provides methods of measuring P450 2C levels in an individual relative to P450 levels in a control population. Such methods entail contacting a sample suspected of containing cytochrome a P450 2C family member from the individual and a substrate of 2C. One then determines the P450 2C levels in the individual relative to P450 2C levels in the control population.